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1.
ACS Appl Mater Interfaces ; 14(1): 138-149, 2022 Jan 12.
Article in English | MEDLINE | ID: covidwho-1574636

ABSTRACT

Highly sensitive, reliable assays with strong multiplexing capability for detecting nucleic acid targets are significantly important for diagnosing various diseases, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The nanomaterial-based assay platforms suffer from several critical issues such as non-specific binding and highly false-positive results. In this paper, to overcome such limitations, we reported sensitive and remarkably reproducible magnetic microparticles (MMPs) and a surface-enhanced Raman scattering (SERS)-based assay using stable silver nanoparticle clusters for detecting viral nucleic acids. The MMP-SERS-based assay exhibited a sensitivity of 1.0 fM, which is superior to the MMP-fluorescence-based assay. In addition, in the presence of anisotropic Ag nanostructures (nanostars and triangular nanoplates), the assay exhibited greatly enhanced sensitivity (10 aM) and excellent signal reproducibility. This assay platform intrinsically eliminated the non-specific binding that occurs in the target detection step, and the controlled formation of stable silver nanoparticle clusters in solution enabled the remarkable reproducibility of the results. These findings indicate that this assay can be employed for future practical bioanalytical applications.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Magnetite Nanoparticles/chemistry , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Humans , Limit of Detection , Metal Nanoparticles/chemistry , RNA, Viral/analysis , RNA, Viral/chemistry , RNA-Dependent RNA Polymerase/genetics , Reproducibility of Results , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Silver/chemistry , Spectrum Analysis, Raman
2.
Cell Rep ; 37(4): 109882, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1525720

ABSTRACT

Remdesivir (RDV), a nucleotide analog with broad-spectrum features, has exhibited effectiveness in COVID-19 treatment. However, the precise working mechanism of RDV when targeting the viral RNA-dependent RNA polymerase (RdRP) has not been fully elucidated. Here, we solve a 3.0-Å structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRP elongation complex (EC) and assess RDV intervention in polymerase elongation phase. Although RDV could induce an "i+3" delayed termination in meta-stable complexes, only pausing and subsequent elongation are observed in the EC. A comparative investigation using an enterovirus RdRP further confirms similar delayed intervention and demonstrates that steric hindrance of the RDV-characteristic 1'-cyano at the -4 position is responsible for the "i+3" intervention, although two representative Flaviviridae RdRPs do not exhibit similar behavior. A comparison of representative viral RdRP catalytic complex structures indicates that the product RNA backbone encounters highly conserved structural elements, highlighting the broad-spectrum intervention potential of 1'-modified nucleotide analogs in anti-RNA virus drug development.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , RNA-Dependent RNA Polymerase/drug effects , SARS-CoV-2/drug effects , Viral Proteins/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , COVID-19/drug therapy , Cryoelectron Microscopy , Humans , RNA, Viral/chemistry , RNA, Viral/drug effects , RNA-Dependent RNA Polymerase/chemistry , SARS-CoV-2/chemistry , Viral Proteins/chemistry , Virus Replication/drug effects
3.
J Endocrinol Invest ; 44(12): 2675-2684, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1504521

ABSTRACT

PURPOSE: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. METHODS: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital. RESULTS: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. CONCLUSION: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.


Subject(s)
COVID-19/diagnosis , Pathology, Molecular/methods , Semen/virology , Adult , Animals , Chlorocebus aethiops , Feasibility Studies , Humans , Male , RNA, Messenger/chemistry , RNA, Viral/chemistry , Real-Time Polymerase Chain Reaction , Reproductive Techniques , Vero Cells
4.
Microb Genom ; 7(11)2021 11.
Article in English | MEDLINE | ID: covidwho-1501253

ABSTRACT

Since the beginning of the SARS-CoV-2 spread in Brazil, few studies have been published analysing the variability of viral genome. Herein, we described the dynamic of SARS-CoV-2 strains circulating in Brazil from May to September 2020, to better understand viral changes that may affect the ongoing pandemic. Our data demonstrate that some of the mutations identified are currently observed in variants of interest and variants of concern, and emphasize the importance of studying previous periods in order to comprehend the emergence of new variants. From 720 SARS-CoV-2 genome sequences, we found few sites under positive selection pressure, such as the D614G (98.5 %) in the spike, that has replaced the old variant; the V1167F in the spike (41 %), identified in the P.2 variant that emerged from Brazil during the period of analysis; and I292T (39 %) in the N protein. There were a few alterations in the UTRs, which was expected, however, our data suggest that the emergence of new variants was not influenced by mutations in UTR regions, since it maintained its conformational structure in most analysed sequences. In phylogenetic analysis, the spread of SARS-CoV-2 from the large urban centres to the countryside during these months could be explained by the flexibilization of social isolation measures and also could be associated with possible new waves of infection. These results allow a better understanding of SARS-CoV-2 strains that have circulated in Brazil, and thus, with relevant infomation, provide the potential viral changes that may have affected and/or contributed to the current and future scenario of the COVID-19 pandemic.


Subject(s)
COVID-19/virology , Genome, Viral , Mutation , SARS-CoV-2/genetics , 5' Untranslated Regions/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Amino Acid Substitution , Brazil/epidemiology , COVID-19/epidemiology , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , Selection, Genetic , Young Adult
5.
PLoS Pathog ; 17(10): e1009726, 2021 10.
Article in English | MEDLINE | ID: covidwho-1484867

ABSTRACT

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.


Subject(s)
Prenylation/physiology , RNA Viruses/drug effects , RNA-Binding Proteins/pharmacology , Virus Replication/physiology , CpG Islands/physiology , HEK293 Cells , HIV-1/physiology , HeLa Cells , Humans , RNA Viruses/physiology , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Binding Motifs/physiology , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , SARS-CoV-2/physiology , Transfection , Virus Replication/drug effects
6.
J Phys Chem Lett ; 12(42): 10277-10283, 2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1469948

ABSTRACT

Guanine quadruplex (G4) structures in the viral genome have a key role in modulating viruses' biological activity. While several DNA G4 structures have been experimentally resolved, RNA G4s are definitely less explored. We report the first calculated G4 structure of the RG-1 RNA sequence of SARS-CoV-2 genome, obtained by using a multiscale approach combining quantum and classical molecular modeling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of the RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.


Subject(s)
COVID-19/genetics , G-Quadruplexes , Genome, Viral , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/virology , Humans , Models, Molecular , Nucleic Acid Conformation
7.
Int J Mol Sci ; 22(20)2021 Oct 12.
Article in English | MEDLINE | ID: covidwho-1463715

ABSTRACT

G-quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of key cellular processes, such as transcription and replication. Since their discovery, G4s have been mainly investigated for their role in cancer and as targets in anticancer therapy. More recently, exploration of the presence and role of G4s in viral genomes has led to the discovery of G4-regulated key viral pathways. In this context, employment of selective G4 ligands has helped to understand the complexity of G4-mediated mechanisms in the viral life cycle, and highlighted the possibility to target viral G4s as an emerging antiviral approach. Research in this field is growing at a fast pace, providing increasing evidence of the antiviral activity of old and new G4 ligands. This review aims to provide a punctual update on the literature on G4 ligands exploited in virology. Different classes of G4 binders are described, with emphasis on possible antiviral applications in emerging diseases, such as the current COVID-19 pandemic. Strengths and weaknesses of G4 targeting in viruses are discussed.


Subject(s)
Antiviral Agents/chemistry , G-Quadruplexes , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , DNA, Viral/chemistry , DNA, Viral/metabolism , Humans , Ligands , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification , Virus Diseases/drug therapy , Virus Diseases/pathology
8.
Science ; 374(6567): eabj3624, 2021 10 29.
Article in English | MEDLINE | ID: covidwho-1440797
9.
Vet Res ; 52(1): 121, 2021 Sep 16.
Article in English | MEDLINE | ID: covidwho-1414142

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.


Subject(s)
COVID-19/pathology , Respiratory System/pathology , SARS-CoV-2/pathogenicity , Animals , Cricetinae , Immunohistochemistry/veterinary , Male , Mesocricetus , Pilot Projects , RNA, Viral/chemistry , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/veterinary , Respiratory System/chemistry , Respiratory System/ultrastructure , Respiratory System/virology , Time Factors , Trachea/pathology , Trachea/ultrastructure , Trachea/virology , Weight Loss
10.
Virus Res ; 305: 198555, 2021 11.
Article in English | MEDLINE | ID: covidwho-1412516

ABSTRACT

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. ß-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly resulting in insufficient inactivation, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 Vaccines/chemistry , Propiolactone/pharmacology , SARS-CoV-2/drug effects , Virion/drug effects , Virus Inactivation/drug effects , Animals , Antigens, Viral/chemistry , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Flocculation/drug effects , Humans , Immune Sera/chemistry , RNA, Viral/chemistry , RNA, Viral/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Vaccines, Inactivated , Vero Cells , Virion/chemistry , Virion/immunology
11.
Sci Signal ; 14(689)2021 06 29.
Article in English | MEDLINE | ID: covidwho-1406596

ABSTRACT

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Subject(s)
COVID-19/virology , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Humans , Manganese/metabolism , Methylation , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Nucleic Acid Conformation , RNA Caps/chemistry , RNA Caps/genetics , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , Signal Transduction , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
12.
Nucleic Acids Res ; 49(18): 10604-10617, 2021 10 11.
Article in English | MEDLINE | ID: covidwho-1406489

ABSTRACT

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.


Subject(s)
Algorithms , RNA, Double-Stranded/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Base Pairing , Base Sequence , COVID-19/prevention & control , Humans , Hydrolysis , RNA Stability , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Viral/genetics , RNA, Viral/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Thermodynamics
14.
PLoS One ; 16(5): e0247626, 2021.
Article in English | MEDLINE | ID: covidwho-1388902

ABSTRACT

SARS-CoV-2 infection in minks has become a serious problem, as the virus may mutate and reinfect humans; some countries have decided to cull minks. Here, the virus sequencing data in minks were analysed and compared to those of human-virus. Although the mink-virus maintained the characteristics of human-virus, some variants rapidly mutated, adapting to minks. Some mink-derived variants infected humans, which accounted for 40% of the total SARS-CoV-2 cases in the Netherlands. These variants appear to be less lethal and infective compared to those in humans. Variants that have mutated further among minks were not found in humans. Such mink-viruses might be suitable for vaccination for humans, such as in the case of the smallpox virus, which is less infective and toxic to humans.


Subject(s)
COVID-19/pathology , Mink/virology , RNA, Viral/chemistry , SARS-CoV-2/genetics , Animals , COVID-19/epidemiology , COVID-19/veterinary , COVID-19/virology , Humans , Mutation , Netherlands/epidemiology , Principal Component Analysis , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Sequence Analysis, RNA
15.
PLoS One ; 16(1): e0245280, 2021.
Article in English | MEDLINE | ID: covidwho-1388897

ABSTRACT

rfaRm is an R package providing a client-side interface for the Rfam database of non-coding RNA and other structured RNA elements. The package facilitates the search of the Rfam database by keywords or sequences, as well as the retrieval of all available information about specific Rfam families, such as member sequences, multiple sequence alignments, secondary structures and covariance models. By providing such programmatic access to the Rfam database, rfaRm enables genomic workflows to incorporate information about non-coding RNA, whose potential cannot be fully exploited just through interactive access to the database. The features of rfaRm are demonstrated by using it to analyze the SARS-CoV-2 genome as an example case.


Subject(s)
RNA, Untranslated/genetics , Sequence Analysis, RNA/methods , Software , Databases, Genetic , Humans , RNA, Untranslated/chemistry , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/genetics
16.
Comput Biol Chem ; 94: 107570, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1385342

ABSTRACT

The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e. g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.


Subject(s)
COVID-19/virology , RNA Folding , RNA, Viral/chemistry , SARS-CoV-2/genetics , Base Sequence , Genome, Viral , RNA, Viral/genetics , RNA, Viral/metabolism
17.
Cell ; 184(1): 184-193.e10, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1385213

ABSTRACT

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Cryoelectron Microscopy , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/chemistry , Viral Replicase Complex Proteins/chemistry , Amino Acid Sequence , Coronavirus/chemistry , Coronavirus/classification , Coronavirus/enzymology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Methyltransferases/metabolism , Models, Molecular , RNA Helicases/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , SARS-CoV-2/enzymology , Sequence Alignment , Transcription, Genetic , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication
18.
Biochem Biophys Res Commun ; 545: 75-80, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1385064

ABSTRACT

Antiviral drug discovery continues to be an essential complement to vaccine development for overcoming the global pandemic caused by SARS-CoV-2. The genomic RNA of SARS-CoV-2 contains structural elements important for viral replication and/or pathogenesis making them potential therapeutic targets. Here we report on the stem-loop II motif, a highly conserved noncoding RNA element. Based on our homology model we determined that the G to U transversion in the SARS-CoV-2 stem-loop II motif (S2MG35U) forms a C-U base-pair isosteric to the C-G base-pair in the early 2000's SARS-CoV (S2M). In addition, chemo-enzymatic probing and molecular dynamics simulations indicate the S2MG35U conformational profile is altered compared to S2M in the apical loop region. We explored S2MG35U as a potential drug target by docking a library of FDA approved drugs. Enzymatic probing of the best docking ligands (aminoglycosides and polymyxins) indicated that polymyxin binding alters the conformational profile and/or secondary structure of the RNA. The SARS-CoV-2 stem-loop II motif conformational differences due to nucleotide transversion and ligand binding are highly significant and provide insight for future drug discovery efforts since the conformation of noncoding RNA elements affects their function.


Subject(s)
RNA, Viral/chemistry , SARS-CoV-2/genetics , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Base Pairing , Binding Sites , Ligands , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Nucleic Acid Conformation , RNA, Untranslated/chemistry , RNA, Untranslated/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism
19.
Angew Chem Int Ed Engl ; 60(21): 11884-11891, 2021 05 17.
Article in English | MEDLINE | ID: covidwho-1384108

ABSTRACT

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Protons , RNA, Viral/analysis , SARS-CoV-2/chemistry , Magnetic Phenomena , RNA, Viral/chemistry
20.
Nat Commun ; 12(1): 1936, 2021 03 29.
Article in English | MEDLINE | ID: covidwho-1387331

ABSTRACT

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Binding Sites , COVID-19/virology , Dimerization , Molecular Dynamics Simulation , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Conformation , Protein Domains
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